The objective of this study is to develop an efficient plasmid transforma- tion system for the gram-positive, pathogenic anaerobe, Clostridium perfringens. The research plan is divided into three specific objectives: 1) to optimize plasmid transformation in C. perfringens 3626B by investigating procedures for efficient formation of protoplasts and cell wall regeneration, 2) to examine the role of cell-wall compartmentalized DNase in C. perfringens DNA uptake, transfer and recovery and 3) to investigate the role of the small, non-conjugative pHB101 plasmid in mediating C. perfringens proteinase activity (lambda toxin) using the gene transfer techniques developed in specific objectives 1 and 2. The effect of glycine on C. perfringens protoplast formation and cell lysis will be examined. Modifications will be made to the protoplasting men- struum and to the cell wall regeneration medium in order to optimize the cell wall regeneration frequency. The role of autolysins in the reversion of C. perfringens cell wall will be examined by a) protease treatment of protoplasts and b) the use of autolysis-deficient mutants. The role of cell-wall compartmentalized DNase in DNA uptake; transfer and recovery will be examined by investigating the effect of various treatments on the frequency of transformation and DNase activity of C. perfringens 3626B protoplasts. The effect of plasmid size, DNA concentration and configuration on the transformation frequency of C. perfringens 3626B protoplasts will also be examined. Liposome-mediated transformation will be utilized to overcome the DNase barrier and optimize the transformation frequency. Finally, efficient PEG-induced or liposome-mediated transformation techniques developed in this proposal will be used to transform C. perfringens 3624A or 3626B lambda toxin minus strains with plasmid pHB101 to definitively demonstrate the coding function of this plasmid. Knowledge gained from this study will provide an increased understanding of the genetic mechanism of pathogenicity of this microorganism. Furthermore, an examination into the role of cell-wall compartmentalized DNase in DNA uptake and transfer, together with the development of a transformation procedure which allows for efficient regeneration of protoplasts to walled cells, should contribute significantly to a basic understanding of gene transfer in the Clostridia.